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Results for “Functional characterization”

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PubMed2003

Molecular characterization, function and regulation of ammonium transporters (Amt) and ammonium-metabolizing enzymes (GS, NADP-GDH) in the ectomycorrhizal fungus Hebeloma cylindrosporum.

External hyphae, which play a key role in nitrogen nutrition of trees, are considered as the absorbing structures of the ectomycorrhizal symbiosis. Here, we have cloned and characterized Hebeloma cylindrosporum AMT1, GLNA and GDHA genes, which encode a third ammonium transporter, a glutamine synthetase and an NADP-dependent glutamate dehydrogenase respectively. Amt1 can fully restore the pseudohyphal growth defect of a Saccharomyces cerevisiae mep2 mutant, and this is the first evidence that a heterologous member of the Mep/Amt family complements this dimorphic change defect. Dixon plots of the inhibition of methylamine uptake by ammonium indicate that Amt1 has a much higher affinity than the two previously characterized members (Amt2 and Amt3) of the Amt/Mep family in H. cylindrosporum. We also identified the intracellular nitrogen pool(s) responsible for the modulation of expression of AMT1, AMT2, AMT3, GDHA and GLNA. In response to exogenously supplied ammonium or glutamine, AMT1, AMT2 and GDHA were downregulated and, therefore, these genes are subjected to nitrogen repression in H. cylindrosporum. Exogenously supplied nitrate failed to induce a downregulation of the five mRNAs after transfer of mycelia from a N-starved condition. Our results demonstrate that glutamine is the main effector for AMT1 and AMT2 repression, whereas GDHA repression is controlled by intracellular ammonium, independently of the intracellular glutamine or glutamate concentration. Ammonium transport activity may be controlled by intracellular NH4+. AMT3 and GLNA are highly expressed but not highly regulated. A model for ammonium assimilation in H. cylindrosporum is presented.

Agaricales↗

PubMed2002

Synthesis, purification and structural and functional characterization of recombinant form of a common genetic variant of human luteinizing hormone.

A common genetic variant (V) of luteinizing hormone (LH), with two mutations (Trp(8)Arg and Ile(15)Thr) and an extra glycosylation consensus site (Asn(13)-Ala-Thr), is associated with abnormalities of reproductive function. To address the molecular basis of the functional differences between V- and wild-type (WT)-LH, recombinant (rec) forms of WT- and V-LH were synthesized in human embryonic kidney (HEK 293) cells. The rec hormones synthesized were rigorously purified employing affinity, immunoaffinity and ion exchange chromatographies (final purity approximately 12 000 IU/mg, 180-fold purification, 28% recovery). Functional properties of the hormone preparations were compared in vitro and in vivo. The molecular size of both rec LHs was 31 kDa, as determined by SDS-PAGE. Although the mutations in V-LHbeta did not significantly affect the affinity of LH receptor (LHR) binding (Kd approximately 0.4 nmol/L), V-LH had higher in vitro biopotency than WT-LH, in terms of mLTC-1 mouse Leydig tumor cell cAMP and progesterone (P) production, and steroidogenic acute regulatory protein (StAR) expression. In addition, in HEK 293 cells expressing the human LHR, V-LH demonstrated 1.8-fold higher response of inositol trisphosphate (IP(3)) production than WT-LH. Furthermore, HEK 293 cells expressing the ElK1 trans-reporting plasmids displayed 2.7-fold greater luciferase response to V-LH than WT-LH, documenting stimulation of the mitogen-activated protein kinase (MAPK) pathway. The in vivo half-life of V-LH was clearly faster (5-9 min) than that of WT-LH (12-22 min) and human chorionic gonadotropin (hCG; 50-70 min), when injected into rat circulation. It is worth noting that analysis by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) demonstrated clear differences in structures of carbohydrate side chains attached to the two forms of rec LHs, including incomplete processing of high mannose glycans (Man(5,8,9)) in V-LH, suggesting different pathways in its intracellular trafficking. Collectively, the present findings provide the molecular basis for the qualitative and quantitative differences in LH action that are observed in carriers of the V-LHbeta allele.

Carbohydrate Conformation↗

PubMed2005

A carotenoid cleavage dioxygenase from Vitis vinifera L.: functional characterization and expression during grape berry development in relation to C13-norisoprenoid accumulation.

A potential Carotenoid Cleavage Dioxygenase (CCD) gene was identified among a Vitis vinifera L. EST collection and a full-length cDNA (VvCCD1) was isolated. Recombinant expression of VvCCD1 confirmed that the gene encoded a functional CCD. Experimental evidence was obtained that VvCCD1 cleaves zeaxanthin symmetrically yielding 3-hydroxy-beta-ionone, a C(13)-norisoprenoidic compound, and a C(14)-dialdehyde. Expression of the gene was studied by real-time PCR at different developmental stages of grape berries from Muscat of Alexandria and Shiraz cultivars. A significant induction of the gene expression approaching véraison was observed in both cultivars. In parallel, the C(13)-norisoprenoid level increased from véraison to maturity in both cultivars.

Carbon Isotopes↗

PubMed1995

Cloning, functional characterization, and mechanism of action of the B-cell-specific transcriptional coactivator OCA-B.

Biochemical purification and cognate cDNA cloning studies have revealed that the previously described transcriptional coactivator OCA-B consists of a 34- or 35-kDa polypeptide with sequence relationships to known coactivators that function by protein-protein interactions. Studies with a recombinant protein have proved that a single OCA-B polypeptide is the main determinant for B-cell-specific activation of immunoglobulin (Ig) promoters and provided additional insights into its mechanism of action. Recombinant OCA-B can function equally well with Oct-1 or Oct-2 on an Ig promoter, but while corresponding POU domains are sufficient for OCA-B interaction, and for octamer-mediated transcription of a histone H2B promoter, an additional Oct-1 or Oct-2 activation domain(s) is necessary for functional synergy with OCA-B. Further studies additional Oct-1 or Oct-2 activation domain(s) is necessary for functional synergy with OCA-B. Further studies show that Ig promoter activation by Oct-1 and OCA-B requires still other general (USA-derived) cofactors and also provide indirect evidence that distinct Oct-interacting cofactors regulate H2B transcription.

Amino Acid Sequence↗

PubMed2004

Functional characterization of mammalian inositol 1,4,5-trisphosphate receptor isoforms.

Inositol 1,4,5-trisphosphate receptors (InsP3R) play a key role in intracellular calcium (Ca2+) signaling. Three mammalian InsP3R isoforms--InsP3R type 1 (InsP3R1), InsP3R type 2 (InsP3R2), and InsP3R type 3 (InsP3R3) are expressed in mammals, but the functional differences between the three mammalian InsP3R isoforms are poorly understood. Here we compared single-channel behavior of the recombinant rat InsP3R1, InsP3R2, and InsP3R3 expressed in Sf9 cells, reconstituted into planar lipid bilayers and recorded with 50 mM Ba2+ as a current carrier. We found that: 1), for all three mammalian InsP3R isoforms the size of the unitary current is 1.9 pA and single-channel conductance is 74-80 pS; 2), in optimal recording conditions the maximal single-channel open probability for all three mammalian InsP3R isoforms is in the range 30-40%; 3), in optimal recording conditions the mean open dwell time for all three mammalian InsP3R isoforms is 7-8 ms, the mean closed dwell time is approximately 10 ms; 4), InsP3R2 has the highest apparent affinity for InsP(3) (0.10 microM), followed by InsP3R1 (0.27 microM), and then by InsP3R3 (0.40 microM); 5), InsP3R1 has a high-affinity (0.13 mM) ATP modulatory site, InsP3R2 gating is ATP independent, and InsP3R3 has a low-affinity (2 mM) ATP modulatory site; 6), ATP modulates InsP3R1 gating in a noncooperative manner (n(Hill) = 1.3); 7), ATP modulates InsP3R3 gating in a highly cooperative manner (n(Hill) = 4.1). Obtained results provide novel information about functional properties of mammalian InsP3R isoforms.

Adenosine Triphosphate↗

PubMed1997

Identification and functional characterization of a K+ channel alpha-subunit with regulatory properties specific to brain.

The physiological diversity of K+ channels mainly depends on the expression of several genes encoding different alpha-subunits. We have cloned a new K+ channel alpha-subunit (Kv2.3r) that is unable to form functional channels on its own but that has a major regulatory function. Kv2.3r can coassemble selectively with other alpha-subunits to form functional heteromultimeric K+ channels with kinetic properties that differ from those of the parent channels. Kv2.3r is expressed exclusively in the brain, being concentrated particularly in neocortical neurons. The functional expression of this regulatory alpha-subunit represents a novel mechanism without precedents in voltage-gated channels, which might contribute to further increase the functional diversity of K+ channels necessary to specify the intrinsic electrical properties of individual neurons.

Amino Acid Sequence↗

PubMed1992

Functional characterization of neurokinin-1 receptors on human U373MG astrocytoma cells.

The neurokinin-1 (NK-1, substance P) receptor belongs to the class of seven transmembrane domain (7-TM) receptors that interact with cellular effector systems via guanine nucleotide binding regulatory proteins (G-proteins). In this study, coupling mechanisms of functional NK-1 receptors endogenously expressed in a human astrocytoma cell line (U373MG) were analyzed. Stimulation with substance P (SP) resulted in 1) a rapid increase in inositol 1,4,5-trisphosphate (IP3) synthesis; 2) a rise in cytosolic free calcium concentration ([Ca2+]i); 3) induction of immediate early gene transcription as monitored by c-fos and c-jun expression; and 4) a significant increase in de novo DNA synthesis. Thus, the functional responses induced by stimulation of NK-1 receptors on U373MG strongly correlate with those observed after treatment of primary astrocytes with SP and make U373MG cells a useful in vitro model system for the analysis of NK-1 receptor function on astrocytes in vivo.

Astrocytoma↗

PubMed2005

Entamoeba histolytica: functional characterization of the -234 to -196 bp promoter region of the multidrug resistance EhPgp1 gene.

The multidrug resistance EhPgp1 gene is constitutively expressed in drug resistant trophozoites from Entamoeba histolytica. It has been demonstrated that two CCAAT/enhancer binding sites located in the EhPgp1 gene promoter control its transcriptional activation. However, functional assays of the 5' end of its promoter showed that region from -234 to -196 bp (38 bp) is also important for the EhPgp1 gene transcription. Here, we demonstrated that in the 38 bp region putative cis-activator sequences are located. In silico analysis showed the presence of GATA1, Gal4, Nit-2, and C/EBP consensus sequences. Additionally, we identified three specific DNA-protein complexes, which were competed by a C/EBP, GATA1, and HOX oligonucleotides. Finally, we partially purified three proteins of 64.4, 56.7, and 27.4 kDa. Further investigations are currently in progress to determine the identity of these nuclear factors and how they are interacting with the EhPgp1 gene promoter.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2002

Molecular cloning and functional characterization of chicken brain tau: isoforms with up to five tandem repeats.

Tau is a major microtubule-associated protein in mammalian brain, where it exists as multiple isoforms that are produced from a single gene by alternative mRNA splicing. Here we present the first report on the structure and function of tau protein from a nonmammalian vertebrate. In the adult chicken brain, five main tau isoforms are expressed. One isoform has three tandem repeats, two isoforms have four repeats each, and two isoforms have five repeats each. Similar to mammalian tau, some chicken tau isoforms contain an amino-terminal insert of 53 amino acids. Unlike mammalian tau, a 34 amino acid insert in the proline-rich region upstream of the repeats is alternatively spliced in chicken tau. It is preceded by a constitutively expressed sequence of 17 amino acids that is absent in tau from human and rodent brains. The expression of chicken tau isoforms and their phosphorylation are developmentally regulated, similar to what has been described in mammalian brain. Functionally, chicken tau isoforms with five repeats have the greatest ability to promote microtubule assembly, followed by isoforms with four and three repeats, respectively. The 34 amino acid insert positively influences both the rate and the extent of microtubule assembly, whereas the 53 amino acid insert only influences the extent of assembly.

Aging↗

PubMed2004

Biochemical and functional characterization of protein kinase CK2 in ascidian Ciona intestinalis oocytes at fertilization. Cloning and sequence analysis of cDNA for alpha and beta subunits.

The ubiquitous and pleiotropic dual specificity protein kinase CK2 has been studied and characterized in many organisms, from yeast to mammals. Generally, the enzyme is composed of two catalytic (alpha and/or alpha') and two regulatory (beta) subunits, forming a differently assembled tetramer. Although prone to controversial interpretation, the function of CK2 has been associated with fundamental biological processes such as signal transduction, cell cycle progression, cell growth, apoptosis, and transcription. Less known is the role of CK2 during meiosis and the early phase of embryogenesis. In this work, we studied CK2 activity during oocyte activation, a process occurring at the end of oocyte maturation and triggered by fertilization. In ascidian Ciona intestinalis, an organism whose complete genome has been published recently, CK2 was constitutively active in unfertilized and fertilized oocytes. The enzymatic activity oscillated through meiosis showing three major peaks: soon after fertilization (metaphase I exit), before metaphase II, and at the exit from metaphase II. Biochemical analysis of CK2 subunit composition in activated oocytes indicated that CK2-alpha was catalytically active as a monomer, independently from its regulatory subunit beta; however, CK2-beta was only detectable in unfertilized oocytes where it was associated with a bona fide identified ascidian mitogen-activated protein kinase. After fertilization, CK2-beta was undetectable, suggesting its rapid degradation. Protein sequence analysis of CK2-alpha and -beta cDNA indicated a high identity compared with vertebrate homologs. In addition, the absence of putative phosphorylation sites for Cdc2 kinase on both alpha and beta subunits suggested an important role for CK2 in regulating meiotic cell cycle in C. intestinalis oocytes.

Amino Acid Sequence↗

PubMed1985

Sézary syndrome: phenotypic and functional characterization of the neoplastic cells.

Phenotypic properties of the neoplastic cells in skin, blood and lymph node specimens from 5 patients with the Sézary syndrome were examined by immuno-enzymatic and -fluorescence labelling of cells and tissue sections with a monoclonal antibody panel. In 3 cases, the in vitro functional properties of the neoplastic cells (isolated from blood specimens) were also analysed using a reverse plaque-forming cell assay. 3 different immunological categories were identified as follows: T-helper/inducer neoplasms (3 patients); T-suppressor/cytotoxic neoplasms (1 patient); and neoplastic T-cells demonstrating characteristics consistent with a concept of their derivation from inducible suppressor T-cells (1 patient). These data provide conclusive evidence that Sézary syndrome is heterogeneous with respect to the immunological characteristics of the neoplastic cells.

Aged↗

PubMed2005

Functional characterization of three naturally occurring single nucleotide polymorphisms in the CES2 gene encoding carboxylesterase 2 (HCE-2).

Twelve single nucleotide polymorphisms (SNPs) in the human CES2 gene, which encodes a carboxylesterase, hCE-2 [human carboxylesterase 2 (EC 3.1.1.1)], have been reported in the Japanese. In this report, we have examined functional alterations of three SNPs, a nonsynonymous SNP (100C>T, R34W), an SNP at the splice acceptor site in intron 8 (IVS8-2A>G), and one newly discovered nonsynonymous SNP (424G>A, V142M). For the two nonsynonymous SNPs, the corresponding variant cDNAs were expressed in COS-1 cells. Both the R34W and V142M variants showed little esterase activities toward the anticancer agent irinotecan and two typical carboxylesterase substrates, p-nitrophenol acetate and 4-methylumbelliferyl acetate, although increased levels of cDNA-mediated protein expression were observed by Western blotting as compared with the wild type. To investigate a possible splicing aberration in IVS8-2A>G, an in vitro splicing assay was utilized and transcripts derived from CES2 gene fragments of the wild type and IVS8-2A>G were compared. Sequence analysis of the cloned transcripts revealed that IVS8-2A>G yielded mostly aberrantly spliced transcripts, including a deleted exon or a 32-bp deletion proximal to the 5' end of exon 9, which resulted in truncated hCE-2 proteins. These results suggested that 100C>T (R34W), 424G>A (V142M), and IVS8-2A>G are functionally deficient SNPs.

Animals↗

PubMed2005

Mapping and functional characterization of the TAF11 interaction with TFIIA.

TFIIA interacts with TFIID via association with TATA binding protein (TBP) and TBP-associated factor 11 (TAF11). We previously identified a mutation in the small subunit of TFIIA (toa2-I27K) that is defective for interaction with TAF11. To further explore the functional link between TFIIA and TAF11, the toa2-I27K allele was utilized in a genetic screen to isolate compensatory mutants in TAF11. Analysis of these compensatory mutants revealed that the interaction between TAF11 and TFIIA involves two distinct regions of TAF11: the highly conserved histone fold domain and the N-terminal region. Cells expressing a TAF11 allele defective for interaction with TFIIA exhibit conditional growth phenotypes and defects in transcription. Moreover, TAF11 imparts changes to both TFIIA-DNA and TBP-DNA contacts in the context of promoter DNA. These alterations appear to enhance the formation and stabilization of the TFIIA-TBP-DNA complex. Taken together, these studies provide essential information regarding the molecular organization of the TAF11-TFIIA interaction and define a mechanistic role for this association in the regulation of gene expression in vivo.

Gene Expression Regulation, Fungal↗

PubMed2005

Structural and functional characterization of polyphenols isolated from acerola (Malpighia emarginata DC.) fruit.

Two anthocyanins, cyanidin-3-alpha-O-rhamnoside (C3R) and pelargonidin-3-alpha-O-rhamnoside (P3R), and quercitrin (quercetin-3-alpha-O-rhamnoside), were isolated from acerola (Malpighia emarginata DC.) fruit. These polyphenols were evaluated based on the functional properties associated with diabetes mellitus or its complications, that is, on the radical scavenging activity and the inhibitory effect on both alpha-glucosidase and advanced glycation end product (AGE) formation. C3R and quercitrin revealed strong radical scavenging activity. While the inhibitory profiles of isolated polyphenols except quercitrin towards alpha-glucosidase activity were low, all polyphenols strongly inhibited AGE formation.

Anthocyanins↗

PubMed1997

Phenotypic and functional characterization of mice that lack the type I receptor for IL-1.

IL-1 alpha and IL-1 beta bind to receptors termed the type I and type II IL-1 receptors. The type I IL-1 receptor is responsible for specific signaling, while the type II IL-1 receptor functions as a nonsignaling decoy receptor. To determine the effect of a defect in IL-1-mediated signaling, mice have been produced with a genetically disrupted type I IL-1 receptor gene. Mice lacking type I IL-1 receptors are of normal vigor and exhibit no overt phenotype. B cells from type I IL-1R-/- mice activated in vitro with anti-IgM do not proliferate in response to IL-1, but do so in response to IL-4. Injection of murine IL-1 alpha does not induce detectable serum IL-6 levels in type I IL-1R-/- mice, but equivalent levels are produced in response to LPS. Type I IL-1R-/- mice have normal serum Ig levels and generate equivalent primary and secondary Ab responses as wild-type mice. In response to LPS, acute phase protein mRNA induction are equivalent in type I IL-1R-/- and wild-type mice. Type I IL-1R-/- mice do not differ from control mice in susceptibility to either a lethal challenge with D-galactosamine plus LPS or high dose LPS. Interestingly, ICE-/-/type I IL-1R-/- double mutant mice are resistant to high dose LPS. Type I IL-1R-/- mice backcrossed to the C57BL/6 background were as equally resistant as wild-type mice to Listeria monocytogenes.

Acute-Phase Proteins↗

PubMed1998

Isolation and functional characterization of cDNA of serum amyloid A-activating factor that binds to the serum amyloid A promoter.

Serum amyloid A (SAA), a plasma protein inducible in response to many inflammatory conditions, is associated with the pathogenesis of several diseases including reactive amyloidosis, rheumatoid arthritis, and atherosclerosis. We have previously reported an element of the SAA promoter, designated SAA-activating sequence (SAS), that is involved in the inflammation-induced SAA expression, and a nuclear factor, SAS-binding factor (SAF), that interacts with the SAS element has been identified previously (A. Ray and B. K. Ray, Mol. Cell. Biol. 16:1584-1594, 1996). To evaluate how SAF is involved in SAA promoter activation, we have investigated structural features and functional characteristics of this transcription factor. Our studies indicate that SAF belongs to a family of transcription factors characterized by the presence of multiple zinc finger motifs of the Cys2-His2 type at the carboxyl end. Of the three cloned SAF cDNAs (SAF-1, SAF-5, and SAF-8), SAF-1 isoform showed a high degree of homology to MAZ/ZF87/Pur-1 protein while SAF-5 and SAF-8 isoforms are unique and are related to SAF-1/MAZ/ZF87/Pur-1 at the zinc finger domains but different elsewhere. Although structurally distinct, all members are capable of activating SAS element-mediated expression and display virtually identical sequence specificities. However, varying levels of expression of members of this gene family were observed in different tissues. Functional activity of SAF is regulated by a posttranslational event as SAF DNA-binding and transactivation abilities are increased by a protein phosphatase inhibitor, okadaic acid, and inhibited by a protein kinase inhibitor, H7. Consistent with this observation, increased DNA binding of the cloned SAF and its hyperphosphorylation, in response to okadaic acid treatment of the transfected cells, were observed. Taken together, our results suggest that, in addition to tissue-specific expression, SAFs, a family of zinc finger transcription factors, undergo a modification by a posttranslational event that confers their SAA promoter-binding activity and transactivation potential.

Amino Acid Sequence↗

PubMed2006

Functional characterization of the NS2B/NS3 protease complex from seven viruses belonging to different groups inside the genus Flavivirus.

The genus Flavivirus, family Flaviviridae, comprises more than 70 viruses. Many of them cause severe, potentially fatal, human diseases. Human vaccines are available for only three viruses and no effective antiviral drug is available. In order to limit the consequences of infections with flaviviruses, a promising approach consists in developing specific compounds that target the virus-encoded NS2B/NS3 protease complex, which is crucial for the viral polyprotein processing. In order to develop such compounds active as antiviral drugs against several flaviviruses, identification of biochemical properties shared by proteases from different viruses is essential. In this work, the functional similarity between the proteases from seven flaviviruses belonging to different major groups was addressed by characterizing their enzymatic properties. For each virus, a catalytically active recombinant protease was designed and expressed as a hexahistidine-tagged protein. Chromogenic and fluorogenic substrates were used to identify optimal conditions for proteolysis. Our study identified important physico-chemical properties shared by all the seven proteases we studied (high pH value requirement for optimal activity, inhibition of substrate processing by salt). However, it also evidenced slight differences in biochemical properties of the flaviviral proteases, which could sustain heterogeneous sensitivity to future inhibitors.

Amino Acid Sequence↗

PubMed2005

Functional characterizations of novWUS involved in novobiocin biosynthesis from Streptomyces spheroides.

NovW, novU, and novS gene products represent dTDP-4-keto-6-deoxy-D-glucose 3,5 epimarase, C-methyltransferase and dTDP-glucose-4-ketoreductase involved in noviose biosynthetic pathway, respectively. We have expressed three genes to elucidate the functions of NovW, NovU, and NovS in Escherichia coli. NovW and NovU catalyze the formation of dTDP-4-keto-6-deoxy-5-C-methyl-L-lyxo-hexose from dTDP-4-keto-6-deoxy-D-glucose. NovS reduces the product formed from the reaction of NovW with dTDP-4-keto-6-deoxy-D-glucose in the presence of NADH to result in dTDP-l-rhamnose. Furthermore, a pathway for the biosynthesis of noviose is proposed.

Base Sequence↗